Nucleic acid hybridization and antibody immunoassay technologies have been developed that permit rapid, sensitive and specific measurements of organic compounds and microorganisms. Recent advances have been directed toward improving the sensitivity and specificity of these assay systems by enhancing the detection or "reporting" of the antigen-antibody complex or nucleic acid hybrid duplex which is formed. Many approaches have been attempted in this regard. One such example is the multiple labeling of an antigen, antibody or nucleic acid probe with an enzyme to produce a nonisotopic and highly sensitive diagnostic test. For example, multiple copies of enzyme can be chemically coupled to a molecule of avidin. The avidin can then bind strongly to biotin, which has been chemically inked to an antibody, antigen or a nucleic acid probe. The result is the presence of multiple copies of enzyme for every antigen-antibody complex or nucleic acid hybrid formed. Another approach used in nucleic acid hybridization assays is the use of multiple enzyme-labeled probes that hybridize to different sequences on the target genome. Whichever approach is used to amplify the biological signal, the result of the assay is usually determined by the development of a distinct color or fluorescence that is read visually or with an instrument.
The detection of specific nucleic acid sequences through the use of hybridization probes is a well established procedure. One commonly used method involves the immobilization of the target polynucleotide sequence on a solid support (e.g., nitrocellulose, diazobenzyloxymethyl cellulose, nylon, etc.). The immobilized nucleic acid is then denatured, if it is double stranded, and subsequently hybridized to a complementary probe. The probe nucleic acid sequence is labeled isotopically, usually with .sup.32 p, or nonisotopically with direct labeling of the polynucleotide sequence with an enzyme or indirectly with a biotin-avidin system.
In contrast to radioisotopically labeled probes, nonisotopic systems offer advantages of safety, relatively low cost, and ease of use- However, enzyme detection often suffers from high background values from the nonspecific adsorption of labeled probes to the solid support. Non-specific adsorption may be reduced with multiple washing steps, which add to the length and difficulty of the procedure.
A different method for detecting a specific polynucleotide sequence involves the displacement of a labeled nucleic acid, according to the method of Vary ,et al., "Nonisotopic Detection Methods for Strand Displacement Assays of Nucleic Acids," Clin. Chem. 32:1696-1701 (1986). A labeled polynucleotide "signal strand" is hybridized with a larger sequence (the "probe strand"), which is, in turn, complementary to the target polynucleotide sequence of interest. Interaction of the target sequence with the signal-probe hybrid results in the displacement of the signal strand from the hybrid. After separating the displaced signal strands from the signal-probe hybrid, the signal strand is measured using an isotopic label such as .sup.32 p or nonisotopic labels such as an enzyme. Such assays are potentially more sensitive because of the reduction of background signal due to nonspecific adsorption.
Two types of enzyme immunoassays are commonly used. The sandwich immunoassay involves the capturing of antigen molecules in a solution by solid phase-bound antibody molecules. A second antibody molecule, which is enzyme-labeled and specific to a different antigenic determinant, is subsequently added to the solid phase-bound antigen-antibody complex. Similarly, the competition immunoassay involves the competition of antigens for antibody binding sites. Enzyme-labeled antigen and unlabeled antigen from the sample (the antigen of interest) compete for binding sites on the solid phase bound antibody. In these cases, the amount of enzyme remaining on the solid support is either proportional, in the first example, or inversely proportional in the second example, to the amount of antigen in the sample.
Attempts at increasing the sensitivity of enzyme immunoassays (EIA) and hybridization assays have frequently focused on increasing the amount of product generated per antigen-antibody complex or hybrid formed by increasing the number of labeled enzyme molecules. Enzyme amplification often results in an increase in false positive reactions due to increased nonspecific adsorption or an increase in false negative reactions due to inhibition of antigen and antibody binding or hybridization by complementary polynucleotide sequences.
Little effort has been directed towards increasing assay sensitivity by enhancing the measurement of the signal or "product" that is generated by the enzyme reacting with the substrate. Frequently, the assay sensitivity is reduced because of a high background-signal. The measurement of extremely low levels of colored or fluorescent enzyme-generated product by an instrument is often compromised by the inherent color or fluorescence of the substrate. This problem can be further exacerbated by the common use of high concentrations of substrate to accommodate a low binding affinity of the enzyme. Background signal can also result from assay and sample components that are colored, fluorescent, luminescent or electrochemically active. In most cases, a positive result is reported only when the enzyme-generated signal is twice the background signal.
In addition to the use of enzymes for detecting immunoreactions and hybridization reactions, little progress has been made for increasing assay sensitivity for detecting free enzymes in a sample as well as enzymes produced by microorganisms. Assays to measure and detect free enzymes and microbial enzymes in a biological sample generally utilize substrates that produce enzyme-generated products that are colored, fluorescent, luminescent or electrochemically active. The sensitivity of these assays is most hindered by a high background signal from sample constituents and assay components including substrate.
One attempt to enhance the measurement of an enzyme-generated product was described by Kiuchi et al. (A Fluorometric Microassay Procedure for Monitoring the Enzymatic Activity of GM1-Ganglioside-B-Galactosidase by Use of High-Performance Liquid Chromatography, 1984, Anal. Biochem. 140:146-151). These investigators utilized a high performance liquid chromatography (HPLC) system to measure the GMi-ganglioside-.beta.-galactosidase activity in crude tissue samples by measuring increased NADH concentration. The biological steps of this procedure, including the incubation of sample with substrate, were conducted in a vessel separate and apart from the HPLC instrument. Following incubation of the substrate and enzyme from the sample, the reaction solution was injected into an HPLC instrument which separated the various assay components. The disadvantage of this procedure is that a conventional HPLC column with a high number of theoretical plates is required to sufficiently separate the components. This means that the separation procedure of Kiuchi et al. is a lengthy procedure and requires the use of an expensive HPLC instrument which is capable of moving fluids through the large column at high pressures, often in excess of 3,000 psi. The column used by Kiuchi et al. had the dimensions of 4 mm.times.300 mm and was packed with reverse phase C18 particles. A column of this type will typically have in excess of 15,000 theoretical plates at optimal linear efficiency.
Wehmeyer et al. (Liquid Chromatography with Electrochemical Detection of Phenol and NADH for Enzyme Immunoassay, 1983, J. Liquid Chromatography 6:2141-56) refers to an enzyme immunoassay procedure with a smaller HPLC column with the dimensions of 50 mm.times.2 mm to separate phenol from other components in the reaction solution. Phenol was generated by the enzymatic cleavage of phenylphosphate. Similar to the procedure at Kiuchi et al., Wehmeyer et al. performed the enzyme immunoreaction in a vessel separate from the HPLC instrument. After sufficient incubation time for the enzyme and substrate in this vessel, the reaction solution was injected into the HPLC instrument. Wehmeyer et al. needed a long HPLC column to accomplish sufficient separation of phenol. The problem with a long HPLC column is an increase in analysis time and the required use of HPLC rated components which can handle high pressure as a result of the use of a long column. Also, extraneous materials in the reaction solution can potentially co-elute with phenol resulting in a significant reduction in, overall assay sensitivity and specificity.
Therefore, there is a need in the art for a method and device to increase the sensitivity of non-isotopic immunoassays and nucleic acid hybridization assays that use enzymes for reporting assay results. Additionally, there is a need in the art for methods for measuring and detecting free enzymes from microorganisms in a sample and from microorganisms in pure culture.